Simple sequence repeats or microsatellites have found widespread usage in the linkage analysis of genetic traits. These sequences are tandem repeats of simple motifs that occur abundantly at random locations throughout the genomes of most eukaryotes. Microsatellites are ideal substrates for PCR due to the fact that they are generally flanked by unique sequence and the overall amplicon size is usually in the region of 100 bases. These sequences display polymorphic variation in the number of repeat units between the flanking sequences. The repeat length polymorphism is not only stable enough to facilitate genetic analysis but is also highly informative for the purpose of linkage analysis. Microsatellites thus serve as ideal markers for the construction of high resolution genetic maps and for the genomic localisation of regions of biological and medical interest.
Current methods for typing the number of repeats at such loci are time consuming and tedious. The number of repeats is determined by PCR product sizing in various electrophoretic separation systems. The PCR products generated upon amplification of genomic DNA may be labelled in a variety of ways. The currently preferred method employs the attachment of a fluorescent moiety to the PCR fragment in order to facilitate detection. A major limitation is the number of samples that can be analysed on a single gel. The current art--using multiple emission wavelength fluors--only allows up to about 10 PCR products to be analysed per gel lane.
In the current art, electrophoretic sizing of PCR products is complicated not only by mobility distortions introduced by the attached fluorescent moieties but also by the exact sequences chosen for the size markers (different sequence compositions for the same fragment length may result in small differences in mobility for some electrophoretic separation systems). Also, addition of non-template directed bases by the polymerase during PCR may lead to multiple fragments appearing in the electropherogram--complicating the subsequent analysis of repeat length considerably.